Protease-activated receptor PAR4 (ZCHEMR2)

ABSTRACT

The present invention relates to polynucleotide and polypeptide molecules for PAR4, a novel member of the protease-activated receptor family. The polypeptides, and polynucleotides encoding them, mediate biological responses and/or cellular signaling in response to proteases. Protease cleavage of PAR4 exposes a PAR4 extracellular amino terminal portion that serves as a ligand for the PAR4 receptor. PAR4 may be used as a target in drug screening, and further used to identify proteinaceous or non-proteinaceous PAR4 agonists and antagonists. The present invention also includes antibodies to the PAR4 polypeptides.

This is a divisional application of application Ser. No. 09/053,866,filed Apr. 1, 1998 now U.S. Pat. No. 6,111,075.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The government may have certain rights in this application by virtue offederal funding under Grant No. 5 R01 HL15919-23 (National Institutes ofHealth).

BACKGROUND OF THE INVENTION

An intriguing question in cell biology relates to the mechanism(s) bywhich proteases activate cells. In recent years, a subfamily of Gprotein-coupled receptors capable of mediating cellular signaling inresponse to proteases has been identified (T. K. H. Vu et al, Cell64:1057–68, 1991; U. B. Rasmussen et al. FEBS Lett. 288:123–28, 1991; S.Nystedt et al., Proc. Natl. Acad. Sci. USA 91:9208–12, 1994; H. Ishiharaet al., Nature 353:674–77, 1997). Members of this unique Gprotein-coupled receptor family include protease-activated receptorsPAR1, PAR2 and PAR3. These receptors are characterized by a tetheredpeptide ligand at the extracellular amino terminus that is generated byminor proteolysis.

The first identified member of this family was the thrombin receptorpresently designated protease-activated receptor 1 (PAR1). Thrombincleaves an amino-terminal extracellular extension of PAR1 to create anew amino terminus that functions as a tethered ligand andintramolecularly activates the receptor (T. K. H. Vu et al, Cell64:1057–68, 1991). PAR2 mediates signaling following minor proteolysisby trypsin or tryptase, but not thrombin (S. Nystedt et al., Proc. Natl.Acad. Sci. USA 91:9208–12, 1994). Knockout of the gene coding for PAR1provided definitive evidence for a second thrombin receptor in mouseplatelets and for tissue-specific roles for different thrombin receptors(A. Connolly et al., Nature 381:516–19, 1996). PAR3 was identifiedrecently as a second thrombin receptor mediates phophatidyl inositol 4,5diphosphate hydrolysis, and was found to be expressed in a variety oftissues (H. Ishihara et al., Nature 353:674–77, 1997). Many otherproteases (such as factor VIIa, factor Xa, factor XIIa, protein C,neutrophil cathepsin G, mast cell tryptase, and plasmin) displaycellular effects. Therefore, additional members of the PAR family areexpected to exist (S. R. Coughlin, Proc. Natl. Acad. Sci. USA91:9200–02, 1994; M. Molino et al., J. Biol. Chem. 272:11133–41, 1997).

The present invention provides an additional member of the PAR family, anovel human protease-activated receptor designated PAR4 (alternativelydesignated ZCHEMR2). The PAR4 polypeptide is an appropriate target fordrug screening, and has other uses that should be apparent to thoseskilled in the art from the teachings herein.

SUMMARY OF THE INVENTION

The present invention provides a novel human protease activated receptorpolypeptide and related compositions and methods.

Within one aspect, the present invention provides an isolatedpolynucleotide encoding a PAR4 polypeptide selected from the groupconsisting of (a) polynucleotide molecules comprising a nucleotidesequence as shown in SEQ ID NO:1 from nucleotide 176 to nucleotide 1330;(b) allelic variants of (a); (c) orthologs of (a); and (d) degeneratenucleotide sequences of (a), (b) or (c). In one embodiment, thepolynucleotide molecules comprise a nucleotide sequence as shown in SEQID NO: 1 from nucleotide 227 to nucleotide 1330. In another embodiment,the polynucleotide molecules comprise a nucleotide sequence as shown inSEQ ID NO: 1 from nucleotide 317 to nucleotide 1330.

Within another aspect, the present invention provides an isolatedpolynucleotide molecule encoding a PAR4 ligand selected from the groupconsisting of (a) polynucleotide molecules comprising a nucleotidesequence as shown in SEQ ID NO: 1 from nucleotide 317 to nucleotide 409;(b) allelic variants of (a); (c) orthologs of (a); and (d) degeneratenucleotide sequences of (a), (b) or (c).

Within yet another aspect, there is provided an expression vectorcomprising the following operably linked elements a transcriptionpromoter; a DNA segment selected from the group consisting of (a)polynucleotide molecules comprising a nucleotide sequence as shown inSEQ ID NO:1 from nucleotide 176 to nucleotide 1330; (b) allelic variantsof (a); (c) orthologs of (a); and (d) degenerate nucleotide sequences of(a), (b) or (c); and a transcription terminator. The present inventionalso provides a cultured cell into which has been introduced suchexpression vector, wherein the cell expresses the PAR4 polypeptide.

Within a further aspect, the invention provides an isolated PAR4polypeptide selected from the group consisting of (a) polypeptidemolecules comprising an amino acid sequence as shown in SEQ ID NO: 2from residue 18 (Gly) to residue 385 (Gln); (b) allelic variants of (a);and (c) orthologs of (a), wherein the PAR4 polypeptide is aprotease-activated receptor.

The invention further provides an isolated PAR4 ligand selected from thegroup consisting of (a) polypeptide molecules comprising an amino acidsequence as shown in SEQ ID NO:2 from residue 48 (Gly) to residue 53(Val); (b) allelic variants of (a); and (c) orthologs of (a), as well asa pharmaceutical composition comprising purified PAR4 ligand incombination with a pharmaceutically acceptable vehicle. Another aspectof the invention provides an antibody that binds to an epitope of a PAR4polypeptide.

These and other aspects of the invention will become evident uponreference to the following detailed description of the invention andattached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The FIGURE depicts the predicted seven transmembrane organization ofPAR4. The signal peptide is located N-terminal of the signal peptidasecleavage site (denoted “S.P.”), and is shaded. The amino terminalpeptide cleaved by thrombin is located between the S.P. cleavage siteand the thrombin cleavage site (denoted “Thrombin”). A 6 amino acidtethered peptide ligand is situated C-terminal of the thrombin cleavagesite and is shaded. The CHD sequence in the second transmembrane loop islocated at the upper right of the second extracellular loop (designatedwith a bar). A potential serine phosphorylation site for protein kinaseC in the sequence SGR (in the third intracellular loop), and a potentialphosphorylation site for protein kinase II in the sequence SPGD (in theC-terminal extracellular domain), are indicated by shading and arrows. Υindicates a potential carbohydrate binding site.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu—Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952–4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204–10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95–107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “complement of a polynucleotide molecule” denotes apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence. For example, thesequence 5′ ATGCACGGG 3′ (SEQ ID NO:13) is complementary to 5′ CCCGTGCAT3′(SEQ ID NO:14).

The term “contig” denotes a polynucleotide that has a contiguous stretchof sequence that is identical or complementary to that of anotherpolynucleotide. Contiguous sequences are said to “overlap” a givenstretch of polynucleotide sequence, either in their entirety or along apartial stretch of the polynucleotide. For example, representativecontigs to the polynucleotide sequence 5′-ATGGTTAGCTT-3′ (SEQ ID NO:15)are 5′-TAGCTTgagtct-3′ (SEQ ID NO:16) and 3′-gtcgacTACCGA-5′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see, for example, Dynan and Tijan, Nature 316:774–78,1985).

An “isolated polypeptide or isolated protein” is a polypeptide orprotein that is found in a condition other than its native environment,such as apart from blood and animal tissue. In a preferred form, theisolated polypeptide is substantially free of other polypeptides,particularly other polypeptides of animal origin. It is preferred toprovide the polypeptides in a highly purified form, i.e. greater than95% pure, more preferably greater than 99% pure. When used in thiscontext, the term “isolated” does not exclude the presence of the samepolypeptide in alternative physical forms, such as dimers oralternatively glycosylated or derivatized forms.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules, it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus, all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain structure (also sometimes referred to as a “multi-peptide”,wherein subunit binding and signal transduction can be functions ofseparate subunits) comprising an extracellular ligand-binding domain andan intracellular effector domain that is typically involved in signaltransduction. Binding of ligand to receptor results in a conformationalchange in the receptor that causes an interaction between the effectordomain and other molecule(s) in the cell. This interaction in turn leadsto an alteration in the metabolism of the cell. Metabolic events thatare linked to receptor-ligand interactions include gene transcription,phosphorylation, dephosphorylation, increases in cyclic AMP production,mobilization of cellular calcium, mobilization of membrane lipids, celladhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids.In general, receptors can be membrane bound, cytosolic or nuclear;monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergicreceptor) or multimeric (e.g., PDGF receptor, growth hormone receptor,IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptorand IL-6 receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

All references cited herein are incorporated by reference in theirentirety.

The present invention is based in part upon the discovery of a novel DNAsequence that encodes a polypeptide having the structure of a seventransmembrane domain protein that features an open reading frame of 385amino acids. This polypeptide, designated PAR4 or ZCHEMR2, has about 33%amino acid sequence identity with PAR1, PAR2 or PAR3. A putative serineprotease cleavage site (R47/G48) was identified within the extracellularamino terminal portion of the polypeptide.

Analysis of the tissue distribution of the mRNA corresponding to thisPAR4 polynucleotide showed that expression was highest in lung,pancreas, thyroid, testis and small intestine. Moderate expression ofPAR4 was observed in prostate, placenta, skeletal muscle, lymph node,adrenal gland, uterus and colon. PAR4 mRNA was also detected in humanplatelets by RT-PCR, but the level of this expression was less than thatof PAR1. No expression of PAR4 was detected in brain, kidney, spinalcord or peripheral blood leukocytes.

The novel PAR4 polypeptides of the present invention were initiallyidentified by querying an EST database for sequences homologous to PAR1,PAR2 and/or PAR3. An EST sequence was identified, and matched a sequenceof the three known PARs in a portion of the fourth transmembrane domain.The deduced amino acid sequence corresponding to this EST sequenceshared 34% identity with the PAR2 amino acid sequence in thetransmembrane region. A full length cDNA clone (4.9 kb) corresponding tothis EST was isolated from a size-selected lymphoma Daudi cell cDNAlibrary.

The nucleotide sequence of PAR4 (ZCHEMR2) is described in SEQ ID NO:1;its deduced amino acid sequence is described in SEQ ID NO:2; and itscorresponding degenerate DNA sequence is described in SEQ ID NO:3. Thepolynucleotide sequence within the full length clone included an openreading frame encoding a 385 amino acid protein (1155 nucleotides, fromnucleotide 176 to nucleotide 1330), including a 17 amino acid signalpeptide (amino acid residues M1 to S17, corresponding to nucleotide 176to nucleotide 226). In addition, SEQ ID NO:1 describes 175 nucleotidesof 5′-untranslated region (nucleotide 1 to nucleotide 175), and a longGC-rich 3′-untranslated region containing several polyadenylationsignals and a poly(A) tail (3565 nucleotides; from nucleotide 1331 tonucleotide 4895).

A hydropathy plot of the amino acid sequence of SEQ ID NO:2 showed thatthe receptor is a member of the seven transmembrane domain receptorfamily. A hydrophobic signal sequence was identified, having a potentialsignal peptidase cleavage site at S17/G18. A putative cleavage site forprotease activation at R47/G48 was also located within the extracellularamino terminus portion of the polypeptide. The extracellular aminoterminus and the intracellular carboxy terminus of PAR4 have little orno amino acid sequence homology to the corresponding regions of thethree known PARs. Further, the protease cleavage site in PAR2 issubstantially different from that in PAR1, PAR2 and PAR3, as shown inTable 1.

TABLE 1 Protease Cleavage Sites in PAR1, PAR2 PAR3 and PAR 4. PAR1TLDPR↓SFLLRNPNDKYEPFWEDEEK (SEQ ID NO:18) (37–61) PAR2SSKGR↓SLIGKVDGTSHVTGKGVTVE (SEQ ID NO:19) (32–56) PAR3 TLPIK↓TFRGAPPNSFEEFPFSALE (SEQ ID NO:20) (34–57) PAR4 LPAPR↓GYPGQVCANDSDTLELPDSS (SEQID NO:21) (28–52) Regions important for fibrinogen anion exosite bindingin thrombin are underlined.

In the second extracellular loop, PAR4 has only three amino acids (CHD)that match the sequence of ITTCHDV (SEQ ID NO:4) that is conserved inPAR1, PAR2 and PAR3. The second extracellular loop is important indetermining specificity of PAR1 from human and X. laevis sources fortheir respective activating peptides (R. E. Gerszten et al., Nature368:548–51, 1994).

The present invention also provides polynucleotide molecules, includingDNA and RNA molecules, that encode the PAR4 polypeptides disclosedherein. Those skilled in the art will readily recognize that, in view ofthe degeneracy of the genetic code, considerable sequence variation ispossible among these polynucleotide molecules. SEQ ID NO:3 is adegenerate DNA sequence that encompasses all DNAs that encode the PAR4polypeptide of SEQ ID NO:2. Those skilled in the art will recognize thatthe degenerate sequence of SEQ ID NO:3 also provides all RNA sequencesencoding SEQ ID NO:2 by substituting U for T. Thus, PAR4polypeptide-encoding polynucleotides comprising nucleotide 1 tonucleotide 1330 of SEQ ID NO:1 and their RNA equivalents arecontemplated by the present invention. Table 2 sets forth the one-lettercodes used within SEQ ID NO:3 to denote degenerate nucleotide positions.“Resolutions” are the nucleotides denoted by a code letter. “Complement”indicates the code for the complementary nucleotide(s). For example, thecode Y denotes either C or T, and its complement R denotes A or G, Abeing complementary to T, and G being complementary to C.

TABLE 2 Nucleotide Resolution Nucleotide Complement A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO:3, encompassing all possiblecodons for a given amino acid, are set forth in Table 3.

TABLE 3 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO:2. Variant sequences can be readily tested forfunctionality as described herein.

It is to be recognized that, according to the present invention, when apolynucleotide is claimed as described herein, it is understood thatwhat is claimed are both the sense strand, the anti-sense strand, andthe DNA as double-stranded having both the sense and anti-sense strandannealed together by their respective hydrogen bonds. Also claimed isthe messenger RNA (mRNA) which encodes the polypeptides of the presentinvention, and which mRNA is encoded by the cDNA described herein.Messenger RNA (mRNA) will encode a polypeptide using the same codons asthose defined herein, with the exception that each thymine nucleotide(T) is replaced by a uracil nucleotide (U).

One of ordinary skill in the art will also appreciate that differentspecies can exhibit “preferential codon usage.” In general, see Granthamet al., Nucl. Acids Res. 8:1893–912, 1980; Haas et al., Curr. Biol.6:315–24, 1996; Wain-Hobson et al., Gene 13:355–64, 1981; Grosjean andFiers, Gene 18:199–209, 1982; Holm, Nucl. Acids Res. 14:3075–87, 1986;Ikemura, J. Mol. Biol. 158:573–97, 1982. As used herein, the term“preferential codon usage” or “preferential codons” is a term of artreferring to protein translation codons that are most frequently used incells of a certain species, thus favoring one or a few representativesof the possible codons encoding each amino acid (See Table 3). Forexample, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG,or ACT, but in mammalian cells ACC is the most commonly used codon; inother species, for example, insect cells, yeast, viruses or bacteria,different Thr codons may be preferential. Preferential codons for aparticular species can be introduced into the polynucleotides of thepresent invention by a variety of methods known in the art. Introductionof preferential codon sequences into recombinant DNA can, for example,enhance production of the protein by making protein translation moreefficient within a particular cell type or species. Therefore, thedegenerate codon sequence disclosed in SEQ ID NO:3 serves as a templatefor optimizing expression of polynucleotides in various cell types andspecies commonly used in the art and disclosed herein. Sequencescontaining preferential codons can be tested and optimized forexpression in various species, and tested for functionality as disclosedherein.

Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO:1,or a sequence complementary thereto, under stringent conditions. Ingeneral, stringent conditions are selected to be about 5° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Typical stringent conditions are those in whichthe salt concentration is up to about 0.03 M at pH 7 and the temperatureis at least about 60° C.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of PAR4 RNA. Such tissues and cells areidentified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA77:5201, 1980), and include a lymphoma Daudi cell line, lung, pancreas,thyroid, testis and small intestine. Total RNA can be prepared usingguanidine HCl extraction followed by isolation by centrifugation in aCsCl gradient (Chirgwin et al., Biochemistry 18:52–94, 1979). Poly (A)⁺RNA is prepared from total RNA using the method of Aviv and Leder (Proc.Natl. Acad. Sci. USA 69:1408–12, 1972). Complementary DNA (cDNA) isprepared from poly(A)⁺ RNA using known methods. In the alternative,genomic DNA can be isolated. Polynucleotides encoding PAR4 polypeptidesare then identified and isolated by, for example, hybridization or PCR.

A full-length clone encoding PAR4 can be obtained by conventionalcloning procedures. Complementary DNA (cDNA) clones are preferred,although for some applications (e.g., expression in transgenic animals)it may be preferable to use a genomic clone, or to modify a cDNA cloneto include at least one genomic intron. Methods for preparing cDNA andgenomic clones are well known and within the level of ordinary skill inthe art, and include the use of the sequence disclosed herein, or partsthereof, for probing or priming a library. Expression libraries can beprobed with antibodies to PAR4, PAR4 fragments, or other specificbinding partners.

The present invention further provides counterpart polypeptides andpolynucleotides from other species (orthologs). These species include,but are not limited to mammalian, avian, amphibian, reptile, fish,insect and other vertebrate and invertebrate species. Of particularinterest are PAR4 polypeptides from other mammalian species, includingmurine, porcine, ovine, bovine, canine, feline, equine, and otherprimate polypeptides. Orthologs of human PAR4 polypeptides can be clonedusing information and compositions provided by the present invention incombination with conventional cloning techniques. For example, a cDNAcan be cloned using mRNA obtained from a tissue or cell type thatexpresses PAR4 as disclosed herein. Suitable sources of mRNA can beidentified by probing Northern blots with probes designed from thesequences disclosed herein. A library is then prepared from mRNA of apositive tissue or cell line. A PAR4-encoding cDNA can then be isolatedby a variety of methods, such as by probing with a complete or partialhuman cDNA or with one or more sets of degenerate probes based on thedisclosed sequences. A cDNA can also be cloned using the polymerasechain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202), using primersdesigned from the representative human PAR4 sequence disclosed herein.Within an additional method, the cDNA library can be used to transformor transfect host cells, and expression of the cDNA of interest can bedetected with an antibody to PAR4 polypeptide. Similar techniques canalso be applied to the isolation of genomic clones.

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NO:1 represents a single allele of human PAR4 and that allelicvariation and alternative splicing are expected to occur. Allelicvariants of this sequence can be cloned by probing cDNA or genomiclibraries from different individuals according to standard procedures.Allelic variants of the DNA sequence shown in SEQ ID NO:1, includingthose containing silent mutations and those in which mutations result inamino acid sequence changes, are within the scope of the presentinvention, as are proteins which are allelic variants of SEQ ID NO:2.cDNAs generated from alternatively spliced mRNAs, which retain theproperties of the PAR4 polypeptides are included within the scope of thepresent invention, as are polypeptides encoded by such cDNAs and mRNAs.Allelic variants and splice variants of these sequences can be cloned byprobing cDNA or genomic libraries from different individuals or tissuesaccording to standard procedures known in the art.

The present invention also provides isolated PAR4 polypeptides that aresubstantially homologous to the polypeptides of SEQ ID NO:2 and theirorthologs. The term “substantially homologous” is used herein to denotepolypeptides having 50%, preferably 60%, more preferably at least 80%,sequence identity to the sequences shown in SEQ ID NO:2 or theirorthologs. Such polypeptides will more preferably be at least 90%identical, and most preferably 95% or more identical to SEQ ID NO:2 orits orthologs. Percent sequence identity is determined by conventionalmethods. See, for example, Altschul et al., Bull. Math. Bio. 48:603–16,1986; and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915–19,1992. Briefly, two amino acid sequences are aligned to optimize thealignment scores using a gap opening penalty of 10, a gap extensionpenalty of 1, and the “blosum 62” scoring matrix of Henikoff andHenikoff (ibid.), as shown in Table 4 (amino acids are indicated by thestandard one-letter codes). The percent identity is then calculated as:

$\frac{\text{Total~~number~~of~~identical~~matches}}{\begin{matrix}\text{[length~~of~~the~~longer~~sequence~~plus~~the~~number~~of~~gaps} \\\text{introduced~~into~~the~~longer~~sequence~~in~~order~~to} \\\text{align~~the~~two~~sequences]}\end{matrix}} \times 100$

TABLE 4 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Sequence identity of polynucleotide molecules is determined by similarmethods using a ratio as disclosed above.

Variant PAR4 polypeptides or substantially homologous PAR4 polypeptidesare characterized as having one or more amino acid substitutions,deletions or additions. These changes are preferably of a minor nature,that is conservative amino acid substitutions (see Table 5) and othersubstitutions that do not significantly affect the folding or activityof the polypeptide; small deletions, typically of one to about 30 aminoacids; and small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue, a small linker peptide of up to about20–25 residues, or an affinity tag. The present invention thus includespolypeptides of from 6 to 410 [385+25] amino acid residues that comprisea sequence that is at least 50%, preferably at least 80%, and morepreferably 90% or more identical to the corresponding region of SEQ IDNO:2. Polypeptides comprising affinity tags can further comprise aproteolytic cleavage site between the PAR4 polypeptide and the affinitytag. Preferred such sites include thrombin cleavage sites and factor Xacleavage sites.

TABLE 5 Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

The present invention further provides a variety of other PAR4 fragmentfusions, and related chimeric or hybrid PAR4 polypeptides or fragments.For example, a PAR4 fragment can be prepared as a fusion to a dimerizingprotein, as disclosed in U.S. Pat. Nos. 5,155,027 and 5,567,584.Preferred dimerizing proteins in this regard include immunoglobulinconstant region domains. Immunoglobulin-PAR4 fragment fusions can beexpressed in genetically engineered cells to produce a variety ofmultimeric PAR4 fragment analogs. Auxiliary domains can be fused to PAR4fragment to target them to specific cells, tissues, or macromolecules(e.g., collagen). For example, a PAR4 fragment could be targeted to apredetermined cell type by fusing a PAR4 fragment to a non-PAR4 moietysuch that the fusion protein specifically binds to a receptor on thesurface of the target cell. In this way, polypeptides and proteins canbe targeted for therapeutic or diagnostic purposes. A PAR4 fragment canbe fused to two or more moieties, such as an affinity tag forpurification and a targeting domain. Fragment fusions can also compriseone or more cleavage sites, particularly between domains. See Tuan etal., Connective Tissue Research 34:1–9, 1996.

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is carried outin a cell-free system comprising an E. coli S30 extract and commerciallyavailable enzymes and other reagents. Proteins are purified bychromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722, 1991; Ellman et al., Meth. Enzymol. 202:301, 1991; Chung etal., Science 259:806–09, 1993; and Chung et al., Proc. Natl. Acad. Sci.USA 90:10145–49, 1993). In a second method, translation is carried outin Xenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991–98, 1996). Within a third method, E. coli cells are culturedin the absence of a natural amino acid that is to be replaced (e.g.,phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470–76, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395–403, 1993).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for PAR4 amino acidresidues.

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244: 1081–85, 1989; Bass et al., Proc. Natl. Acad.Sci. USA 88:4498–502, 1991). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity asdisclosed below to identify amino acid residues that are critical to theactivity of the molecule. See also, Hilton et al., J. Biol. Chem.271:4699–708, 1996. Sites of ligand-receptor oragonist/antagonist-receptor interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., Science 255:306–12,1992; Smith et al., J. Mol. Biol. 224:899–904, 1992; Wlodaver et al.,FEBS Lett. 309:59–64, 1992. The identities of essential amino acids canalso be inferred from analysis of homologies with related PAR familymembers.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53–57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152–56, 1989). Briefly, these referencesdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832–37, 1991; Ladneret al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Neret al., DNA 7:127, 1988).

Variants of the disclosed PAR4 DNA and polypeptide sequences can begenerated through DNA shuffling, as disclosed by Stemmer, Nature370:389–91, 1994; Stemmer, Proc. Natl. Acad. Sci. USA 91:10747–51, 1994;and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated byin vitro homologous recombination by random fragmentation of a parentDNA, followed by reassembly using PCR, resulting in randomly introducedpoint mutations. This technique can be modified by using a family ofparent DNAs, such as allelic variants or DNAs from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay, provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode active polypeptides (e.g., those activated byproteases; those that mediate a biological response in the presence ofproteases; those that stimulate the PAR4 receptor itself) can berecovered from the host cells and rapidly sequenced using modernequipment. These methods allow the rapid determination of the importanceof individual amino acid residues in a polypeptide of interest, and canbe applied to polypeptides of unknown structure.

Using the methods discussed herein, one of ordinary skill in the art canidentify and/or prepare a variety of polypeptide fragments or variantsof SEQ ID NO:2 that retain the PAR family properties of the wild-typePAR4 protein. Such polypeptides may include a complete extracellularamino terminus portion; an extracellular amino terminus portioncorresponding to amino acid residues G18 through G48, or to amino acidresidues G18 through R78, or to amino acid residues G48 through R78; anextracellular portion linked to one or more of the seven transmembranedomains of PAR4; and the like.

For any PAR4 polypeptide, including variants and fusion proteins, one ofordinary skill in the art can readily generate a fully degeneratepolynucleotide sequence encoding that variant using the information setforth in Tables 2 and 3, above.

The PAR4 polypeptides of the present invention, including full-lengthpolypeptides, biologically active fragments, and fusion polypeptides,can be produced in genetically engineered host cells according toconventional techniques. Suitable host cells are those cell types thatcan be transformed or transfected with exogenous DNA and grown inculture, and include bacteria, fungal cells, and cultured highereukaryotic cells. Eukaryotic cells, particularly cultured cells ofmulticellular organisms, are preferred. Techniques for manipulatingcloned DNA molecules and introducing exogenous DNA into a variety ofhost cells are disclosed by Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989; and Ausubel et al., eds., Current Protocolsin Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

In general, a DNA sequence encoding a PAR4 polypeptide or a portionthereof is operably linked to other genetic elements required for itsexpression, generally including a transcription promoter and terminator,within an expression vector. The vector will also commonly contain oneor more selectable markers and one or more origins of replication,although those skilled in the art will recognize that within certainsystems selectable markers may be provided on separate vectors, andreplication of the exogenous DNA may be provided by integration into thehost cell genome. Selection of promoters, terminators, selectablemarkers, vectors and other elements is a matter of routine design withinthe level of ordinary skill in the art. Many such elements are describedin the literature and are available through commercial suppliers.

To direct a PAR4 polypeptide or fragment into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of the native PAR4polypeptide, or may be derived from another secreted protein (e.g.,t-PA) or synthesized de novo. The secretory signal sequence is operablylinked to the PAR4 DNA sequence, i.e., the two sequences are joined inthe correct reading frame and positioned to direct the newly synthesizedpolypeptide into the secretory pathway of the host cell. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe polypeptide of interest, although certain secretory signal sequencesmay be positioned elsewhere in the DNA sequence of interest (see, e.g.,Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.5,143,830).

Alternatively, the secretory signal sequence contained in thepolypeptides of the present invention is used to direct otherpolypeptides into the secretory pathway. The present invention providesfor such fusion polypeptides. A signal fusion polypeptide can be madewherein a secretory signal sequence derived from amino acid residues M1to S17 of SEQ ID NO:2 is operably linked to another polypeptide usingmethods known in the art and disclosed herein. The secretory signalsequence contained in the fusion polypeptides of the present inventionis preferably fused amino-terminally to a second peptide to direct theadditional peptide into the secretory pathway. Such constructs havenumerous applications known in the art. For example, these novelsecretory signal sequence fusion constructs can direct the secretion ofan active component of a normally non-secreted protein, such as areceptor. Such fusions may be used in vivo or in vitro to directpeptides through the secretory pathway.

Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841–45, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993), and viral vectors (Miller and Rosman, BioTechniques 7:980–90,1989; Wang and Finer, Nature Med. 2:714–16, 1996). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59–72, 1977) and Chinese hamsterovary (e.g., CHO-K₁; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Rockville, Md. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins, such as CD4, CD8, Class I MHC, placental alkaline phosphatase,may be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plantcells, insect cells and avian cells. The use of Agrobacterium rhizogenesas a vector for expressing genes in plant cells has been reviewed bySinkar et al., J. Biosci. (Bangalore) 11:47–58, 1987. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication WO94/06463. Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV). DNA encoding the PAR4 polypeptide is inserted into thebaculoviral genome in place of the AcNPV polyhedrin gene coding sequenceby one of two methods. The first is the traditional method of homologousDNA recombination between wild-type AcNPV and a transfer vectorcontaining the PAR4 polynucleotide flanked by AcNPV sequences. Suitableinsect cells, e.g., SF9 cells, are infected with wild-type AcNPV andtransfected with a transfer vector comprising a PAR4 polynucleotideoperably linked to an AcNPV polyhedrin gene promoter, terminator, andflanking sequences. See, L. A. King and R. D. Possee, The BaculovirusExpression System: A Laboratory Guide, London, Chapman & Hall; D. R.O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual,New York, Oxford University Press., 1994; and, C. D. Richardson, ed.,Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa,N.J., Humana Press, 1995. Natural recombination within an insect cellwill result in a recombinant baculovirus which contains PAR4 driven bythe polyhedrin promoter. Recombinant viral stocks are made by methodscommonly used in the art.

The second method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (V. A. Luckow et al., J.Virol. 67:4566–79, 1993). This system is sold in the Bac-to-Bac kit(Life Technologies, Rockville, Md.). This system utilizes a transfervector, pFastBacl™ (Life Technologies), containing a Tn7 transposon tomove the DNA encoding the PAR4 polypeptide into a baculovirus genomemaintained in E. coli as a large plasmid called a “bacmid.” ThepFastBacl™ transfer vector utilizes the AcNPV polyhedrin promoter todrive the expression of the gene of interest, in this case PAR4.However, pFastBacl™ can be modified to a considerable degree. Thepolyhedrin promoter can be removed and substituted with the baculovirusbasic protein promoter (also known as Pcor, p6.9 or MP promoter) whichis expressed earlier in the baculovirus infection, and has been shown tobe advantageous for expressing secreted proteins. See, M. S.Hill-Perkins and R. D. Possee, J. Gen. Virol. 71:971–76, 1990; B. C.Bonning et al., J. Gen. Virol. 75:1551–56, 1994; and G. D. Chazenbalkand B. Rapoport, J. Biol. Chem. 270:1543–49, 1995. In such transfervector constructs, a short or long version of the basic protein promotercan be used. Moreover, transfer vectors can be constructed which replacethe native PAR4 secretory signal sequences with secretory signalsequences derived from insect proteins. For example, a secretory signalsequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin(Invitrogen, Carlsbad, Calif.), or baculovirus gp67 (PharMingen, SanDiego, Calif.) can be used in constructs to replace the native PAR4secretory signal sequence. In addition, transfer vectors can include anin-frame fusion with DNA encoding an epitope tag at the C- or N-terminusof the expressed PAR4 polypeptide, for example, a Glu—Glu epitope tag(T. Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952–54, 1985).Using a technique known in the art, a transfer vector containing PAR4 istransformed into E. coli, and screened for bacmids which contain aninterrupted lacZ gene indicative of recombinant baculovirus. The bacmidDNA containing the recombinant baculovirus genome is isolated, usingcommon techniques, and used to transfect Spodoptera frugiperda cells,e.g., Sf9 cells. Recombinant virus that expresses PAR4 is subsequentlyproduced. Recombinant viral stocks are made by methods commonly used theart.

The recombinant virus is used to infect host cells, typically a cellline derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High Five™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media include Sf900 II™ (Life Technologies) or ESF921™(Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRHBiosciences, Lenexa, Kans.) or Express FiveO™ (Life Technologies) forthe T. ni cells. The cells are grown up from an inoculation density ofapproximately 2–5×10⁵ cells to a density of 1–2×10⁶ cells, at which timea recombinant viral stock is added at a multiplicity of infection (MOI)of 0.1 to 10, more typically near 3. The recombinant virus-infectedcells typically produce the recombinant PAR4 polypeptide at 12–72 hourspost-infection and secrete it with varying efficiency into the medium.The culture is usually harvested 48 hours post-infection. Centrifugationis used to separate the cells from the medium (supernatant). Thesupernatant containing the PAR4 polypeptide is filtered throughmicropore filters, usually 0.45 μm pore size. Procedures used aregenerally described in available laboratory manuals (L. A. King and R.D. Possee, ibid.; D. R. O'Reilly et al., ibid.; C. D. Richardson,ibid.). Subsequent purification of the PAR4 polypeptide from thesupernatant can be achieved using methods described herein.

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein Saccharonyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459–65, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533.

The use of Pichia methanolica as host for the production of recombinantproteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, it is preferred that thepromoter and terminator in the plasmid be that of a P. methanolica gene,such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Otheruseful promoters include those of the dihydroxyacetone synthase (DHAS),formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitateintegration of the DNA into the host chromosome, it is preferred to havethe entire expression segment of the plasmid flanked at both ends byhost DNA sequences. A preferred selectable marker for use in Pichiamethanolica is a P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), whichallows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, it is preferred to use host cells in which bothmethanol utilization genes (AUG1 and AUG2) are deleted. For productionof secreted proteins, host cells deficient in vacuolar protease genes(PEP4 and PRB1) are preferred. Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. It is preferred to transform P.methanolica cells by electroporation using an exponentially decaying,pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (τ) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.). When expressing a PAR4polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

It is preferred to purify PAR4 polypeptide fragments or fusions(particularly those that function as PAR4 agonists or antagonists) to≧80% purity, more preferably to ≧90% purity, even more preferably ≧95%purity, and particularly preferred is a pharmaceutically pure state,that is greater than 99.9% pure with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. Preferably, a purified PAR4polypeptide fragment or fusion is substantially free of otherpolypeptides, particularly other polypeptides of animal origin.

Expressed recombinant PAR4 polypeptide fragments, PAR4 fragment fusions,or PAR4 fragment chimeras or hybrids can be purified using fractionationand/or conventional purification methods and media. Ammonium sulfateprecipitation and acid or chaotrope extraction may be used forfractionation of samples. Exemplary purification steps may includehydroxyapatite, size exclusion, FPLC and reverse-phase high performanceliquid chromatography. Suitable chromatographic media includederivatized dextrans, agarose, cellulose, polyacrylamide, specialtysilicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.Exemplary chromatographic media include those media derivatized withphenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties. Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodiimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Methods for binding polypeptides to support mediaare well known in the art. Selection of a particular method is a matterof routine design and is determined in part by the properties of thechosen support. See, for example, Affinity Chromatography: Principles &Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

The PAR4 polypeptide fragments, PAR4 fragment fusions or PAR4 fragmentchimeric or hybrid polypeptides of the present invention can be isolatedby exploitation of PAR family properties. For example, immobilized metalion adsorption (IMAC) chromatography can be used to purify PAR4polypeptides or fragments that comprise a polyhistidine tag. Briefly, agel is first charged with divalent metal ions to form a chelate(Sulkowski, Trends in Biochem. 3:1–7, 1985). Histidine-rich or -taggedproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (Methodsin Enzymol., Vol. 182, “Guide to Protein Purification”, M. Deutscher,(ed.), Acad. Press, San Diego, 1990, pp. 529–39). Within additionalembodiments of the invention, a fusion of the polypeptide or fragment ofinterest and an affinity tag (e.g., maltose-binding protein, animmunoglobulin domain) may be constructed to facilitate purification.

Components of the PAR4 polypeptide may be combined with other Gprotein-coupled receptor components to form chimeric or hybrid Gprotein-coupled receptors. Alternatively, such hybrid or chimericreceptors may include a component of PAR4 from one species and a secondcomponent of PAR4 from another species (see, for example, U.S. Pat. No.5,284,746). More specifically, using regions or domains of the inventivePAR4 protein or fragments thereof in combination with those of otherhuman PAR family proteins or heterologous PAR proteins (Sambrook et al.,ibid.; Altschul et al., ibid.; Picard, Curr. Opin. Biology 5:511–15,1994, and references therein), hybrid or chimeric PAR4 polypeptides orfragments may be obtained through recombinant means (or in the case offragments, may be synthesized). Construction of these polypeptidesallows the determination of the biological importance of larger domainsor regions in a polypeptide of interest. Such hybrids may modulatereaction kinetics or binding, may constrict or expand the substratespecificity, or may alter tissue and cellular localization of apolypeptide, and can be applied to polypeptides of unknown structure.For G protein-coupled receptors, the chimeric or hybrid polypeptides maybe less than full length (for instance, may include none, one or moretransmembrane domains; may include only extracellular portions; and thelike).

Fusion proteins can be prepared by methods known to those skilled in theart by preparing each component of the fusion protein and chemicallyconjugating them. Alternatively, a polynucleotide encoding bothcomponents of the fusion protein in the proper reading frame can begenerated using known techniques and expressed by the methods describedherein. For example, part or all of a domain(s) conferring a biologicalfunction may be swapped between the PAR4 polypeptide or fragment of thepresent invention with the functionally equivalent domain(s) fromanother family member, such as PAR1, PAR2 or PAR3. Such domains include,but are not limited to, the secretory signal sequence, the extracellularN-terminal domain, an extracellular loop, a transmembrane region, anintracellular loop, or the intracellular C-terminal domain. Such fusionproteins would be expected to have a biological and functional profilethat is the same or similar to polypeptides of the present invention orother known G protein-coupled receptor and/or PAR family proteins,depending on the fusion constructed. Moreover, such fusion proteins mayexhibit other properties, as disclosed herein.

PAR4 polypeptides or fragments thereof may also be prepared throughchemical synthesis. PAR4 polypeptides or fragments may be monomers ormultimers; glycosylated or non-glycosylated; pegylated or non-pegylated;and may or may not include an initial methionine amino acid residue.

The activity of molecules of the present invention can be measured usinga variety of assays that measure cellular activation or responses(including platelet activation, adhesion or aggregation), signalingevents, ligand binding or receptor agonism or antagonism. Of particularinterest are assays involving phosphoinositide hydrolysis; mobilizationof intracellular calcium; modification of ligand with active siteinhibitors; mutation of ligand active site residues; ligand antagonists;affinity tag release following proteolysis; and proteasesubstrate/cleavage product determinations. Such assays are well known inthe art. For a general reference, see T. K. H. Vu et al., Cell64:1057–68, 1991; or H. Ishihara et al., Nature 386:502–06, 1997.

Proteins of the present invention are useful for studying the effects ofligand-receptor interactions on cellular activation and responses invitro and in vivo. In addition, the PAR4 polypeptide, fragment orchimeric polypeptide of the present invention may be useful in screeningfor receptor agonists and antagonists. PAR4 activities can be measuredin vitro using cultured cells transfected with the PAR4 polypeptide, orin vivo by administering soluble PAR4 fragments (for instance, portionsof the N-terminal extracellular region) or PAR4 fusion polypeptides ofthe claimed invention to the appropriate animal model.

An alternative in vivo approach for assaying proteins or fragments ofthe present invention involves viral delivery systems. Exemplary virusesfor this purpose include adenovirus, herpesvirus, vaccinia virus andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acid (for a review, see T. C. Becker et al., Meth.Cell Biol. 43:161–89, 1994; and J. T. Douglas and D. T. Curiel, Science& Medicine 4:44–53, 1997). The adenovirus system offers severaladvantages: adenovirus can (i) accommodate relatively large DNA inserts;(ii) be grown to high-titer; (iii) infect a broad range of mammaliancell types; and (iv) be used with a large number of available vectorscontaining different promoters. Also, because adenoviruses are stable inthe bloodstream, they can be administered by intravenous injection.

By deleting portions of the adenovirus genome, larger inserts (up to 7kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. In an exemplary system, theessential E1 gene has been deleted from the viral vector, and the viruswill not replicate unless the E1 gene is provided by the host cell (thehuman 293 cell line is exemplary). When intravenously administered tointact animals, adenovirus primarily targets the liver. If theadenoviral delivery system has an E1 gene deletion, the virus cannotreplicate in the host cells. However, the host's tissue (e.g., liver)will express and process (and, if a secretory signal sequence ispresent, secrete) the heterologous protein. Secreted proteins will enterthe circulation in the highly vascularized liver, and effects on theinfected animal can be determined.

The adenovirus system can also be used for protein production in vitro.By culturing adenovirus-infected non-293 cells under conditions wherethe cells are not rapidly dividing, the cells can produce proteins forextended periods of time. For instance, BHK cells are grown toconfluence in cell factories, then exposed to the adenoviral vectorencoding the secreted protein of interest. The cells are then grownunder serum-free conditions, which allows infected cells to survive forseveral weeks without significant cell division. Alternatively,adenovirus vector infected 293S cells can be grown in suspension cultureat relatively high cell density to produce significant amounts ofprotein (see Garnier et al., Cytotechnol. 15:145–55, 1994). With eitherprotocol, an expressed, secreted heterologous protein can be repeatedlyisolated from the cell culture supernatant. Within the infected 293Scell production protocol, non-secreted proteins may also be effectivelyobtained.

PAR4 agonists and antagonists have enormous potential in both in vitroand in vivo applications. Compounds identified as PAR4 agonists areuseful for up-regulating cellular responses and physiology; PAR4antagonists are useful for down-regulating these same activities. Inaddition, the PAR4 polypeptides and fragments may be used to dissect theeffects of thrombin (a serine protease) or other activating proteases inthe clotting pathway from the effects of thrombin or other activatingproteases at the cellular level. Further, PAR4 agonist compounds areuseful as components of defined cell culture media for growth of cellsexpressing PAR4 and stimulated by protease cleavage and activation ofPAR4. PAR4 fragments or agonists may be used alone or in combinationwith other cytokines, hormones and the like to replace serum that iscommonly used in cell culture. PAR4 agonists are thus useful inspecifically promoting the proliferation and/or differentiation ofplatelets; in mediating inflammatory events, responses to vascularinjury, chemotaxis or mitogenesis; and in promoting production of growthfactors.

PAR4 antagonists are also useful as research reagents for characterizingsites of ligand-receptor interaction. Antibodies directed against PAR4polypeptides and fragments may also serve as useful antagonists for invitro and in vivo studies and administration. More specifically,anti-PAR4 antibodies or PAR4 antagonists may selectively inhibit thecellular effects of thrombin or other activating proteases, whileleaving the clotting pathway fully responsive to thrombin. PAR4antagonists may also be useful for down-regulating biological responsesor activities of cells that overproduce PAR4 or that exhibit increasedintracellular signaling in response to PAR4 stimulation. Thisdown-regulation may be particularly useful for prophylaxis or treatmentof recipients suffering from a disease or syndrome wherein responsivecells (such as platelets) are overproduced or are abnormallyup-regulated. If the PAR4 antagonist is capable of being targeted toand/or localized in specific tissues or organs (such as with fusionpolypeptides having a targeting component), selective decreases incellular activities or responses may be obtained. Soluble PAR4extracellular domains may also be useful as antagonists.

PAR4 agonists and antagonists may be proteinaceous or non-proteinaceous,and may include peptidic and non-peptidic agents (including ribozymes),small molecules and mimetics. PAR4 agonists and antagonists may also beuseful in determining the specificity, activities and distribution ofother PAR family members, as well as in examining the roles played byintracellular signaling components (such as the variety of G proteinspresent in cells) with respect to these PAR family members (and, morebroadly, with respect to G protein-coupled receptor family members).

PAR4 activation may be studied by determining phosphoinositidehydrolysis after protease stimulation. Site-directed mutagenesis isadvantageously used to evaluate protease cleavage (activation) sites inPAR4 polypeptides. Synthetic peptides derived from the unmasked aminoterminus of PAR4 following protease cleavage are also useful in studyingPAR4 activation. Intracellular phosphorylation sites can be examined fortheir involvement in termination of signaling by PAR4. An epitope-taggedPAR4 assay also provides information about cleavage and activation ofPAR4.

Mammalian cells transfected with PAR4 constructs are useful systems forstudying activating peptides, agonists and antagonists of PAR4. A PAR4transfected cell is used to screen for ligands for the receptor, as wellas agonists and antagonists of the natural ligand. To summarize thisapproach, a cDNA or gene encoding the receptor is combined with othergenetic elements required for its expression (e.g., a transcriptionpromoter), and the resulting expression vector is inserted into a hostcell. Cells that express the DNA and produce functional receptors areselected and used within a variety of screening systems.

Cells expressing functional PAR4 are used within screening assays. Avariety of suitable assays are known in the art. These assays are basedon the detection of a biological response in a target cell. An increasein metabolism above a control value indicates a test compound thatmodulates PAR4 activity or responses. One such assay is a cellproliferation assay. Cells are cultured in the presence or absence of atest compound, and cell proliferation is detected by, for example,measuring incorporation of tritiated thymidine or by colorimetric assaybased on the metabolic breakdown of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)(Mosman, J. Immunol. Meth. 65:55–63, 1983). An additional assay methodinvolves measuring the effect of a test compound on receptor (+) cells,containing the receptor of interest on their cell surface, and receptor(−) cells, those which do not express the receptor of interest. Thesecells can be engineered to express a reporter gene. The reporter gene islinked to a promoter element or response element that is responsive tothe receptor-linked pathway, and the assay detects activation oftranscription of the reporter gene. Suitable response elements includecyclic AMP response elements (CRE), hormone response elements (HRE),insulin response elements (IRE) (Nasrin et al., Proc. Natl. Acad. Sci.USA 87:5273–77, 1990), and serum response elements (SRE) (Shaw et al.,Cell 56: 563–72, 1989). Cyclic AMP response elements are reviewed inRoestler et al., J. Biol. Chem. 263 (19):9063–66; 1988; and Habener,Molec. Endocrinol. 4(8):1087–94; 1990. Hormone response elements arereviewed in Beato, Cell 56:335–44; 1989. A preferred promoter element inthis regard is a serum response element, or SRE (see, e.g., Shaw et al.,Cell 56:563–72, 1989). A preferred such reporter gene is a luciferasegene (de Wet et al., Mol. Cell. Biol. 7:725, 1987). Expression of theluciferase gene is detected by luminescence using methods known in theart (e.g., Baumgartner et al., J. Biol. Chem. 269:29094–101, 1994;Schenborn and Goiffin, Promega Notes 41:11, 1993). Luciferase activityassay kits are commercially available from, for example, Promega Corp.,Madison, Wis. Target cell lines of this type can be used to screenlibraries of chemicals, cell-conditioned culture media, fungal broths,soil samples, water samples, and the like. Assays of this type willdetect compounds that directly block PAR4 ligand binding, as well ascompounds that block processes in the cellular pathway subsequent toreceptor-ligand binding. In the alternative, compounds or other samplescan be tested for direct blocking of PAR4 binding using moieties taggedwith a detectable label (e.g., ¹²⁵I, biotin, horseradish peroxidase,FITC, and the like). Within assays of this type, the ability of a testsample to inhibit the activation PAR4 is indicative of inhibitoryactivity, which can be confirmed through secondary assays. The abilityof a test sample to stimulate PAR4 activity may also be determined andconfirmed through secondary assays.

An assay system that uses a ligand-binding receptor (or an antibody, onemember of a complement/anti-complement pair) or a binding fragmentthereof, and a commercially available biosensor instrument (BIAcore,Pharmacia Biosensor, Piscataway, N.J.) may be advantageously employed.Such receptor, antibody, member of a complement/anti-complement pair orfragment is immobilized onto the surface of a receptor chip. Use of thisinstrument is disclosed by Karlsson, J. Immunol. Methods 145:229–40,1991; and Cunningham and Wells, J. Mol. Biol. 234:554–63, 1993. Areceptor, antibody, member or fragment is covalently attached, usingamine or sulfhydryl chemistry, to dextran fibers that are attached togold film within the flow cell. A test sample is passed through thecell. If a ligand, epitope, or opposite member of thecomplement/anti-complement pair is present in the sample, it will bindto the immobilized receptor, antibody or member, respectively, causing achange in the refractive index of the medium, which is detected as achange in surface plasmon resonance of the gold film. This system allowsthe determination of on- and off-rates, from which binding affinity canbe calculated, and assessment of stoichiometry of binding.

Ligand-binding receptor polypeptides can also be used within other assaysystems known in the art. Such systems include Scatchard analysis fordetermination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:660–72, 1949) and calorimetric assays (Cunningham et al., Science253:545–48, 1991; Cunningham et al., Science 245:821–25, 1991).

PAR4 polypeptides can also be used to prepare antibodies thatspecifically bind to PAR4 epitopes, peptides or polypeptides. The PAR4polypeptide or a fragment thereof serves as an antigen (immunogen) toinoculate an animal and elicit an immune response. Suitable antigenswould be the PAR4 polypeptide encoded by SEQ ID NO:2 from amino acidnumber G18 to amino acid number R78, or from amino acid number G48 toamino acid number R78, or from amino acid number C54 to amino acidnumber R78. Alternatively, polypeptides corresponding to any PAR4extracellular loop may be suitable antigens. Antibodies generated fromthis immune response can be isolated and purified as described herein.Methods for preparing and isolating polyclonal and monoclonal antibodiesare well known in the art. See, for example, Current Protocols inImmunology, Cooligan, et al. (eds.), National Institutes of Health, JohnWiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; andHurrell, J. G. R., ed., Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press, Inc., Boca Raton, Fla., 1982.

As would be evident to one of ordinary skill in the art, polyclonalantibodies can be generated from inoculating a variety of warm-bloodedanimals such as horses, cows, goats, sheep, dogs, chickens, rabbits,mice, and rats with a PAR4 polypeptide or a fragment thereof. Theimmunogenicity of a PAR4 polypeptide or fragment may be increasedthrough the use of an adjuvant, such as alum (aluminum hydroxide) orFreund's complete or incomplete adjuvant. Polypeptides useful forimmunization also include fusion polypeptides, such as fusions of PAR4or a portion thereof with an immunoglobulin polypeptide or with maltosebinding protein. The polypeptide immunogen may be a full-length moleculeor a portion thereof. If the polypeptide portion is “hapten-like”, suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingnon-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced.

Alternative techniques for generating or selecting antibodies usefulherein include in vitro exposure of lymphocytes to PAR4 protein orpeptide, and selection of antibody display libraries in phage or similarvectors (for instance, through use of immobilized or labeled PAR4protein or peptide). Genes encoding polypeptides having potential PAR4polypeptide binding domains can be obtained by screening random peptidelibraries displayed on phage (phage display) or on bacteria, such as E.coli. Nucleotide sequences encoding the polypeptides can be obtained ina number of ways, such as through random mutagenesis and randompolynucleotide synthesis. These random peptide display libraries can beused to screen for peptides which interact with a known target which canbe a protein or polypeptide, such as a ligand or receptor, a biologicalor synthetic macromolecule, or organic or inorganic substances.Techniques for creating and screening such random peptide displaylibraries are known in the art (Ladner et al., U.S. Pat. No. 5,223,409;Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S. Pat. No.5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) and random peptidedisplay libraries and kits for screening such libraries are availablecommercially, for instance from Clontech (Palo Alto, Calif.), InvitrogenInc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.) andPharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Random peptidedisplay libraries can be screened using the PAR4 sequences disclosedherein to identify proteins which bind to PAR4. These “binding proteins”which interact with PAR4 polypeptides can be used for tagging cells; forisolating homolog polypeptides by affinity purification; they can bedirectly or indirectly conjugated to drugs, toxins, radionuclides andthe like. These binding proteins can also be used in analytical methodssuch as for screening expression libraries and neutralizing activity.The binding proteins can also be used for diagnostic assays fordetermining circulating levels of polypeptides; for detecting orquantitating soluble polypeptides as marker of underlying pathology ordisease. These binding proteins can also act as PAR4 “antagonists” toblock PAR4 binding and signal transduction in vitro and in vivo. Theseanti-PAR4 binding proteins would be useful for inhibiting cellularresponses to protease-activated PAR4.

Antibodies are determined to be specifically binding if: 1) they exhibita threshold level of binding activity, and/or 2) they do notsignificantly cross-react with related polypeptide molecules. First,antibodies herein specifically bind if they bind to a PAR4 polypeptide,peptide or epitope with a binding affinity (K_(a)) of 10⁶ M⁻¹ orgreater, preferably 10⁷ M⁻¹ or greater, more preferably 10⁸ M⁻¹ orgreater, and most preferably 10⁹ M⁻¹ or greater. The binding affinity ofan antibody can be readily determined by one of ordinary skill in theart, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad.Sci. 51: 660–672, 1949).

Second, antibodies are determined to specifically bind if they do notsignificantly cross-react with related polypeptides. Antibodies do notsignificantly cross-react with related polypeptide molecules, forexample, if they detect PAR4 but not known related polypeptides using astandard Western blot analysis (Ausubel et al., ibid.). Examples ofknown related polypeptides are orthologs, proteins from the same speciesthat are members of a protein family (e.g. PARs), PAR4 polypeptides, andnon-human PAR4. Moreover, antibodies may be “screened against” knownrelated polypeptides to isolate a population that specifically binds tothe inventive polypeptides. For example, antibodies raised to PAR4 areadsorbed to related polypeptides adhered to insoluble matrix; antibodiesspecific to PAR4 will flow through the matrix under the proper bufferconditions. Such screening allows isolation of polyclonal and monoclonalantibodies non-crossreactive to closely related polypeptides(Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988; Current Protocols in Immunology,Cooligan, et al. (eds.), National Institutes of Health, John Wiley andSons, Inc., 1995). Screening and isolation of specific antibodies iswell known in the art. See, Fundamental Immunology, Paul (eds.), RavenPress, 1993; Getzoff et al., Adv. in Immunol. 43:1–98, 1988; MonoclonalAntibodies: Principles and Practice, Goding, J. W. (eds.), AcademicPress Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2:67–101, 1984.

A variety of assays known to those skilled in the art can be utilized todetect antibodies which specifically bind to PAR4 proteins or peptides.Exemplary assays are described in detail in Antibodies: A LaboratoryManual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press,1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutant PAR4protein, polypeptide or fragment.

Antibodies to PAR4 may be used for tagging cells that express PAR4; forisolating PAR4 or PAR4 fragments by affinity purification; fordiagnostic assays for determining circulating levels of PAR4polypeptides or fragments; for detecting or quantitating soluble PAR4 asmarker of underlying pathology or disease; in analytical methodsemploying FACS; for screening expression libraries; for generatinganti-idiotypic antibodies; and as neutralizing antibodies or asantagonists to block PAR4 protease-activated activities in vitro and invivo. Suitable direct tags or labels include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like; indirect tags or labels mayfeature use of biotin-avidin or other complement/anti-complement pairsas intermediates. Antibodies herein may also be directly or indirectlyconjugated to drugs, toxins, radionuclides and the like, and theseconjugates used for in vivo diagnostic or therapeutic applications.Moreover, antibodies to PAR4 or fragments thereof may be used in vitroto detect denatured PAR4 or fragments thereof in assays, for example,Western Blots or other assays known in the art.

Suitable detectable molecules may be directly or indirectly attached tothe polypeptide or antibody, and include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like. Suitable cytotoxic moleculesmay be directly or indirectly attached to the polypeptide or antibody,and include bacterial or plant toxins (for instance, diphtheria toxin,Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeuticradionuclides, such as iodine-131, rhenium-188 or yttrium-90 (eitherdirectly attached to the polypeptide or antibody, or indirectly attachedthrough means of a chelating moiety, for instance). Polypeptides orantibodies may also be conjugated to cytotoxic drugs, such asadriamycin. For indirect attachment of a detectable or cytotoxicmolecule, the detectable or cytotoxic molecule can be conjugated with amember of a complementary/ anticomplementary pair, where the othermember is bound to the polypeptide or antibody portion. For thesepurposes, biotin/streptavidin is an exemplarycomplementary/anticomplementary pair.

In another embodiment, polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues).Alternatively, if the polypeptide has multiple functional domains (i.e.,an activation domain or a ligand binding domain, plus a targetingdomain), a fusion protein including only the targeting domain may besuitable for directing a detectable molecule, a cytotoxic molecule or acomplementary molecule to a cell or tissue type of interest. Ininstances where the domain only fusion protein includes a complementarymolecule, the anti-complementary molecule can be conjugated to adetectable or cytotoxic molecule. Such domain-complementary moleculefusion proteins thus represent a generic targeting vehicle forcell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

In another embodiment, PAR4-cytokine fusion proteins orantibody-cytokine fusion proteins can be used for enhancing in vivokilling of target tissues (for example, blood and bone marrow cancers),if the PAR4 polypeptide or fragment, or the anti-PAR4 antibody, targetsthe hyperproliferative blood or bone marrow cell (see, generally,Hornick et al., Blood 89:4437–47, 1997). This reference described fusionproteins that enable targeting of a cytokine to a desired site ofaction, thereby providing an elevated local concentration of cytokine.Suitable PAR4 polypeptides or fragments or anti-PAR4 antibodies targetan undesirable cell or tissue (i.e., a tumor or a leukemia), and thefused cytokine mediated improved target cell lysis by effector cells.Suitable cytokines for this purpose include interleukin 2 andgranulocyte-macrophage colony-stimulating factor (GM-CSF), for instance.

In yet another embodiment, if the PAR4 polypeptide or anti-PAR4 antibodytargets vascular cells or tissues, such polypeptide or antibody may beconjugated with a radionuclide, and particularly with a beta-emittingradionuclide, to reduce restenosis. Such therapeutic approach poses lessdanger to clinicians who administer the radioactive therapy. Forinstance, iridium-192 impregnated ribbons placed into stented vessels ofpatients until the required radiation dose was delivered showeddecreased tissue growth in the vessel and greater luminal diameter thanthe control group, which received placebo ribbons. Further,revascularisation and stent thrombosis were significantly lower in thetreatment group. Similar results are predicted with targeting of abioactive conjugate containing a radionuclide, as described herein.

The bioactive polypeptide or antibody conjugates described herein can bedelivered intravenously, intraarterially or intraductally, or may beintroduced locally at the intended site of action.

Polynucleotides encoding PAR4 polypeptides or fragments are usefulwithin gene therapy applications where it is desired to increase orinhibit PAR4 activity. If a mammal has a mutated or absent PAR4 gene,the PAR4 gene can be introduced into the cells of the mammal. In oneembodiment, a gene encoding a PAR4 polypeptide or fragment is introducedin vivo in a viral vector. Such vectors include an attenuated ordefective DNA virus, such as, but not limited to, herpes simplex virus(HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus,adeno-associated virus (AAV), and the like. Defective viruses, whichentirely or almost entirely lack viral genes, are preferred. A defectivevirus is not infective after introduction into a cell. Use of defectiveviral vectors allows for administration to cells in a specific,localized area, without concern that the vector can infect other cells.Examples of particular vectors include, but are not limited to, adefective herpes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec.Cell. Neurosci. 2:320–30, 1991); an attenuated adenovirus vector, suchas the vector described by Stratford-Perricaudet et al., J. Clin.Invest. 90:626–30, 1992; and a defective adeno-associated virus vector(Samulski et al., J. Virol. 61:3096–101, 1987; Samulski et al., J.Virol. 63:3822–28, 1989).

In another embodiment, a PAR4 gene can be introduced in a retroviralvector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346;Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol.62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263; InternationalPatent Publication No. WO 95/07358, published Mar. 16, 1995 by Doughertyet al.; and Kuo et al., Blood 82:845, 1993. Alternatively, the vectorcan be introduced by lipofection in vivo using liposomes. Syntheticcationic lipids can be used to prepare liposomes for in vivotransfection of a gene encoding a marker (Felgner et al., Proc. Natl.Acad. Sci. USA 84:7413–17, 1987; Mackey et al., Proc. Natl. Acad. Sci.USA 85:8027–31, 1988). The use of lipofection to introduce exogenousgenes into specific organs in vivo has certain practical advantages.Molecular targeting of liposomes to specific cells represents one areaof benefit. More particularly, directing transfection to particularcells represents one area of benefit. For instance, directingtransfection to particular cell types would be particularly advantageousin a tissue with cellular heterogeneity, such as the pancreas, liver,kidney, and brain. Lipids may be chemically coupled to other moleculesfor the purpose of targeting. Targeted peptides (e.g., hormones orneurotransmitters), proteins such as antibodies, or non-peptidemolecules can be coupled to liposomes chemically.

It is possible to remove the target cells from the body; to introducethe vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963–67, 1992; Wu et al., J. Biol. Chem. 263:14621–24, 1988.

Antisense methodology can be used to inhibit PAR4 gene or fragmenttranscription, such as to inhibit cell proliferation in vivo.Polynucleotides that are complementary to a segment of a PAR4-encodingpolynucleotide (e.g., a polynucleotide as set froth in SEQ ID NO:1) aredesigned to bind to PAR4-encoding mRNA and to inhibit translation ofsuch mRNA. Such antisense polynucleotides are used to inhibit expressionof PAR4 polypeptide-encoding genes in cell culture or in a subject.

The present invention also provides reagents which will find use indiagnostic applications. For example, the PAR4 gene, a probe comprisingPAR4 DNA or RNA or a subsequence thereof can be used to determine if thePAR4 gene is present on a particular chromosome, or if a mutation hasoccurred. Detectable chromosomal aberrations at the PAR4 gene locusinclude, but are not limited to, aneuploidy, gene copy number changes,insertions, deletions, restriction site changes and rearrangements. Suchaberrations can be detected using polynucleotides of the presentinvention by employing molecular genetic techniques, such as restrictionfragment length polymorphism (RFLP) analysis, short tandem repeat (STR)analysis employing PCR techniques, and other genetic linkage analysistechniques known in the art (Sambrook et al., ibid.; Ausubel et al.,ibid.; Marian, Chest 108:255–65, 1995).

Transgenic mice, engineered to express the PAR4 gene, and mice thatexhibit a complete absence of PAR4 gene function, referred to as“knockout mice” (Snouwaert et al., Science 257:1083, 1992), may also begenerated (Lowell et al., Nature 366:740–42, 1993). These mice may beemployed to study the PAR4 gene and the protein encoded thereby in an invivo system.

Radiation hybrid mapping is a somatic cell genetic technique developedfor constructing high-resolution, contiguous maps of mammalianchromosomes (Cox et al., Science 250:245–50, 1990). Partial or fullknowledge of a gene's sequence allows one to design PCR primers suitablefor use with chromosomal radiation hybrid mapping panels. Radiationhybrid mapping panels are commercially available which cover the entirehuman genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RHPanel (Research Genetics, Inc., Huntsville, Ala.). These panels enablerapid, PCR-based chromosomal localizations and ordering of genes,sequence-tagged sites (STSs), and other nonpolymorphic and polymorphicmarkers within a region of interest. This includes establishing directlyproportional physical distances between newly discovered genes ofinterest and previously mapped markers. The precise knowledge of agene's position can be useful for a number of purposes, including: 1)determining if a sequence is part of an existing contig and obtainingadditional surrounding genetic sequences in various forms, such as YACs,BACs or cDNA clones; 2) providing a possible candidate gene for aninheritable disease which shows linkage to the same chromosomal region;and 3) cross-referencing model organisms, such as mouse, which may aidin determining what function a particular gene might have.

Sequence tagged sites (STSs) can also be used independently forchromosomal localization. An STS is a DNA sequence that is unique in thehuman genome and can be used as a reference point for a particularchromosome or region of a chromosome. An STS is defined by a pair ofoligonucleotide primers that are used in a polymerase chain reaction tospecifically detect this site in the presence of all other genomicsequences. Since STSs are based solely on DNA sequence they can becompletely described within an electronic database, for example,Database of Sequence Tagged Sites (dbSTS), GenBank, (National Center forBiological Information, National Institutes of Health, Bethesda, Md.http://www.ncbi.nlm.nih.gov), and can be searched with a gene sequenceof interest for the mapping data contained within these short genomiclandmark STS sequences.

For pharmaceutical use, PAR4 fragments that stimulate or inhibit PAR4activation are formulated for parenteral, particularly intravenous orsubcutaneous, delivery according to conventional methods. Intravenousadministration will be by bolus injection or infusion over a typicalperiod of one to several hours. In general, pharmaceutical formulationswill include a PAR4 fragment in combination with a pharmaceuticallyacceptable vehicle, such as saline, buffered saline, 5% dextrose inwater or the like. Formulations may further include one or moreexcipients, preservatives, solubilizers, buffering agents, albumin toprevent protein loss on vial surfaces, etc. Methods of formulation arewell known in the art and are disclosed, for example, in Remington: TheScience and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. Therapeutic doses will generally be in therange of 0.1 to 100 μg/kg of patient weight per day, preferably 0.5–20μg/kg per day, with the exact dose determined by the clinician accordingto accepted standards, taking into account the nature and severity ofthe condition to be treated, patient traits, etc. Determination of doseis within the level of ordinary skill in the art. The proteins may beadministered for acute treatment, over one week or less, often over aperiod of one to three days or may be used in chronic treatment, overseveral months or years. In general, a therapeutically effective amountof a PAR4 fragment is an amount sufficient to produce a clinicallysignificant change in unwanted cellular activation or responsiveness.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 PAR4 Polynucleotide and Polypeptide

A search of various databases was conducted to identify ESTs withhomology to the three known protease-activated receptors (PAR1, PAR2 andPAR3). One EST sequence was identified that showed homology to the threeprotease-activated receptors in the fourth transmembrane domain. Moreparticularly, the deduced amino acid sequence corresponding to this ESTnucleotide sequence shared 34% identity with PAR2 in the transmembraneregion.

A size-selected lymphoma Daudi cell line cDNA library containing insertsgreater than about 2 kb was then screened, using a 600 bp DNA probederived from the EST sequence. The DNA probe, corresponding tonucleotides 818–1391 of SEQ ID NO:1, was prepared by PCR amplificationusing Daudi cell cDNA as a template. Screening of the cDNA library wascarried out by standard filter hybridization techniques with radioactiveDNA probes labeled by random priming (Prime-it kit, Stratagene, LaJolla, Calif.). cDNA inserts were sequenced on both strands by thedideoxy chain termination method (F. Sanger et al., Proc. Natl. Acad.Sci. USA 74:5463–67, 1977) using the Sequenase Kit from US Biochemicals(Cleveland, Ohio).

A full-length cDNA clone (4.9 kb) was identified, sequenced on bothstrands, and designated as protease-activated receptor 4 (PAR4). The DNAsequence revealed a 5′-untranslated region (nucleotides 1–175 of SEQ IDNO:1), an open reading frame encoding a 385 amino acid protein(nucleotides 176–1330 of SEQ ID NO:1), and a long GC-rich3′-untranslated region containing several polyadenylation signals and apoly(A) tail (nucleotides 1331–4895 of SEQ ID NO:1).

A hydropathy plot of the amino-acid sequence of PAR4 revealed that thereceptor was a member of the seven transmembrane domain receptor family,as illustrated in FIG. 1. A hydrophobic signal sequence with a potentialsignal peptidase cleavage site was present at S17/G18. A putativecleavage site for protease activation at R47/G48 was also present withinthe extracellular amino terminus. Alignment of the PAR4 amino acidsequence with the three other known protease-activated receptorsindicated that PAR4 was a member of the protease-activated receptorfamily, with about 33% overall amino acid sequence identity with PAR1,PAR2, or PAR3. However, the extracellular amino terminus andintracellular carboxy terminus of PAR4 have little or no amino acidsequence similarity to the corresponding regions in the other familymembers. The protease cleavage site in PAR4 differs substantially fromthat in PAR1 and PAR3. In the second extracellular loop, PAR4 has onlythree amino acids (CHD) that match the sequence of ITTCHDV (SEQ ID NO:4)that is conserved in PAR1, PAR2, and PAR3.

Example 2 Activation of PAR4 by Thrombin and Trypsin

The similarity in sequence between PAR4 and the other protease-activatedreceptors suggested that PAR4 should be activated by anarginine-specific serine protease. For comparative purposes, PAR1protein was prepared. Briefly, the cDNA coding for PAR1 was isolatedfrom a placental cDNA library by PCR. The PAR1 DNA sequence obtained wasessentially identical to that previously reported, except fornucleotides 711–712 (CG→GC) and nucleotides 1091–1092 (CG→GC). Thesedifferences resulted in a change of V→L at amino acid residue 238 and achange of S→C at amino acid residue 364, respectively. These amino acidchanges were confirmed by sequence analysis of the corresponding regionsin the genomic DNA coding for PAR1.

COS cells were transiently transfected with PAR4 cDNA, and examined forresponses to thrombin and trypsin. Briefly, for the phosphoinositidehydrolysis assay, COS-7 cells were grown in Dulbecco's modified Eagle'smedium (DMEM; Gibco/BRL, Gaithersburg, Md.) with 10% fetal bovine serum(FBS). Cells were plated at 3.5×10⁵/35-mm plate one day beforetransfection. Two μg of DNA were transfected using 12 μl oflipofectAMINE (Gibco/BRL) for 5 h. The cells were incubated overnight inDMEM with 10% FBS, and then split into triplicate 35-mm wells.Forty-eight hours after transfection, the cells were loaded with 2μCi/ml [³H]myo-inositol (Amersham, Arlington Heights, Ill.) inserum-free DMEM and incubated overnight at 37° C. Cells were washed andtreated with 20 mM LiCl in DMEM, with or without protease or peptideactivators added at various concentrations. Cells were then incubatedfor 2 h at 37° C. and extracted with 750 μl of 20 mM formic acid for 30min on ice. The inositol mono-, bis-, and trisphosphates were purifiedthrough a one ml AG 1-X8 anion-exchange resin (Bio-Rad, Hercules,Calif.) (T. Nanevicz et al., J. Biol. Chem. 271:702–06, 1996), andquantitated by scintillation counting. In each hydrolysis assay, surfaceexpression levels of receptors were determined in triplicate in parallelcultures.

The PAR4-transfected COS cells did respond to thrombin or trypsinaddition (100 nM), resulting in phosphatidylinositol 4,5 diphosphatehydrolysis. This response was comparable to the thrombin-stimulatedactivation of PAR1. Gamma-thrombin that lacks a fibrinogen-bindingexosite (T. J. Rydel et al., J. Biol. Chem. 269:22000–06, 1994)(EnzymeResearch Laboratories, Inc., South Bend, Ind.) was as effective asα-thrombin in the activation of PAR4. This is in contrast to theactivation of PAR1 and PAR3, where γ-thrombin is much less potent thanα-thrombin. This difference in activation is probably due to thepresence of an additional thrombin binding site within the aminoterminal region of PAR1 and PAR3. The thrombin-stimulatedphosphoinositide hydrolysis with PAR4 was dose-dependent, with ahalf-maximal concentration (EC50) for thrombin and trypsin of 5 nM. Thisdose level was much higher than that for PAR1 and PAR3 (about 0.2 nM).

Other arginine/lysine-specific serine proteases, including factor VIIa,IXa, XIa, urokinase, or plasmin, had little or no activity against PAR4.Small effects, however, were observed with factor Xa at highconcentrations (100 nM). Chymotrypsin and elastase failed to activatePAR4.

Site-directed mutagenesis was employed to evaluate the importance of theputative cleavage site at R47/G48 in PAR4 activation. A cDNA encodingPAR4 with a single amino acid substitution of Ala for Arg at residue 47was transiently expressed in COS cells. The putative cleavage sitemutant (R47A) failed to respond to either thrombin or trypsin. Incontrast, a mutation of Arg at residue 68 in the extracellularamino-terminal region (R68A) had no effect on PAR4 activation bythrombin or trypsin in the phosphatidylinositol 4,5 diphosphatehydrolysis assay. Thus, the putative protease cleavage site of R47/G48in PAR4 was critical for receptor activation.

Example 3 Epitope-tagged PAR4 Assay

Surface expression of wild-type and mutant PAR4 polypeptide wasdetermined using specific binding of monoclonal antibody M1 (EastmanKodak Company, Scientific Imaging Systems, New Haven Conn.) directed ata FLAG epitope inserted at PAR4's amino terminus. The cDNA employed forthe epitope-tagged PAR4 assay was prepared analogous toFLAG-epitope-tagged PAR1 with an amino terminus sequence ofMDSKGSSQKGSRLLLLLVVSNLLLCQGVVS↓DYKDDDDKLE-GG (SEQ ID NO:5). Thissequence represents the bovine prolactin signal peptide, the putativesignal peptidase site (↓), the FLAG epitope DYKDDDDK (SEQ ID NO:6), anda junction of LE providing a XhoI cloning site (H. Ishihara et al.,Nature 386:502–06, 1997). This sequence (SEQ ID NO:5) was fused to G18in PAR4. Receptor cDNAs were subcloned into the mammalian expressionvector pZP-7. Receptor expression on the COS cell surface was measuredas specific binding of monoclonal antibody M1 to the FLAG epitope at theamino terminus of PAR4 (see K. Ishii et al., J. Biol. Chem. 268:9780–86,1993).

Briefly, transfected COS cells were split into 24-well plates (Falcon,Becton Dickinson Labware Company, Lincoln Park, N.J.) at 1×10⁵cells/well. One day later, cells were washed with Dulbecco's modifiedEagle's medium (DMEM) containing 10 mM Hepes (pH 7.4), 50 mM Tris-HCl,and 1 mM CaCl₂. Cells were thereafter exposed to various proteases forselected times at 37° C., then fixed with 4% paraformaldehyde in 150 mMsodium chloride, 10 mM sodium phosphate (pH 7.0), 1 mM calcium chloride(phosphate-buffered saline, PBS), 50 mM Tris-HCl for 5 min on ice.Plates were washed twice with PBS, and then incubated with primarymonoclonal anti-FLAG antibody M1 (0.5 μg/ml) inDMEM/Hepes/Tris-HCl/CaCl₂/bovine serum albumin (BSA, 1 mg/ml) for 1 h atroom temperature. Plates were washed with PBS and incubated withhorseradish peroxidase (HRP)-conjugated goat-anti-mouse second antibody(Bio-Rad; 1:1,000 dilution) in DMEM/Hepes/Tris-HCl/CaCl₂/BSA for 30 minat room temperature. After additional washing with PBS, plates weredeveloped with the HRP chromogenic substrate2,2′-azino-di[3-ethyl-benzthiazoline-6-sulfonic acid] (Bio-Rad). OD₄₁₅was read after 5–10 min. Antibody binding data are expressed as specificbinding (total minus nonspecific binding, with nonspecific being definedas the level of binding seen on untransfected control COS cells).

Example 4 Protease Receptor Activating Peptide

The protease-activated receptor family has been shown to be activated bya peptide derived from the amino terminus of the receptor protein.Accordingly, a hexapeptide (GYPGQV; SEQ ID NO:7), corresponding to theamino terminus of PAR4 that is unmasked following cleavage at R47/G48,was tested for its ability to stimulate COS cells expressing PAR4. Thispeptide readily activated both wild-type and mutant PAR4 (R47A) at 500μM, whereas thrombin and trypsin only activate the wild-type PAR4. COScells with no transfected DNA failed to respond to the activatingpeptide under the same conditions. The maximal response of cellsexpressing PAR4 to the activating peptide was comparable to the maximalresponse to thrombin or trypsin. The activating peptide (SFLLRN; SEQ IDNO:8) from PAR1 showed no activity toward PAR4 when tested at aconcentration effective for PAR1 activation. The EC50 of PAR4 activatingpeptide was about 100 μM, which is substantially higher than that of theactivating peptide for PAR1. The high EC50 for the activating peptidefor PAR4, as compared to thrombin or trypsin, clearly reflects thedifference between a built-in tethered ligand and a ligand in freesolution.

Example 5 Potential Intracellular Phosphorylation Sites

Since the termination of the signaling of PAR4 may occur byphosphorylation (analogous to the β-adrenergic receptor; see K. Ishii etal., J. Biol. Chem. 269:1125–1130, 1994), the intracellular regions ofPAR4 were examined for potential phosphorylation sites. A serine residueis present in the third intracellular loop of PAR4 that could bephosphorylated by protein kinase C, while another serine residue ispresent in the carboxy terminal region that could be phosphorylated bycasein kinase II (FIG. 1). Accordingly, the termination of PAR4signaling may be similar to that for other seven transmembranereceptors.

Example 6 Tissue Distribution of PAR4

The tissue distribution of PAR4 was examined by Northern blot analysis.Briefly, three human multiple-tissue blots with 2 μg mRNA in each lane(ClonTech, Palo Alto, Calif.) were hybridized with a [³²P]-labeled 166bp PCR product generated from human lymph node cDNA with PCR4 specificprimers, 5′-TGGCACTGCCCCTGACACTGCA-3′ (SEQ ID NO:9) and5′-CCCGTAGCACAGCAGCATGG-3′ (SEQ ID NO:10). Hybridization to humanβ-actin mRNA was used as a control for variation in abundance. The blotswere hybridized overnight in ExpressHyb (ClonTech) and washed at 50° C.in 0.1×SSC, 0.1% SDS, followed by exposure to X-ray film. Northern blotanalysis of mRNA from 23 different tissues showed that the PAR4 gene wasexpressed in most of the tissues tested, with especially high levels inlung, pancreas, thyroid, testis, and small intestine. Moderateexpression was also detected in prostate, placenta, skeletal muscle,lymph node, adrenal gland, uterus, and colon. No PAR4 expression wasdetected in brain, kidney, spinal cord, and peripheral blood leukocytes.The PAR4 mRNA was also detected in human platelets by RT-PCR, althoughthe expression of PAR4 was much less than that of PAR1.

Example 7 Chromosomal Localization of PAR4

The Human Genetic Mutant Cell Repository Human/Rodent Somatic CellHybrid Mapping Panel Number 2 (National Institute of General MedicalSciences, Coriell Institute of Medical Research) was used with PCRamplification to identify the somatic hybrid that contained the humanPAR4 gene (R. E. Kuestner et al., Mol. Pharm. 46:246–55, 1994). PAR4specific oligonucleotide primers (sense, 5′-GGTGCCCGCCCTCTATGG-3′ (SEQID NO:11), and anti-sense, 5′-TCGCGAGGTTCATCAGCA-3′ (SEQ ID NO:12)) wereused for the PCR amplification. Subchromosomal mapping of the PAR4 genewas carried out using the commercially available version of the StanfordG3 Radiation Hybrid Mapping Panel (Research Genetics, Inc., Huntsville,Ala.). The Stanford G3 RH Panel contains PCR-amplifiable DNAs from eachof 83 radiation hybrid clones of the whole human genome, plus twocontrol DNAs (the RM donor and the A3 recipient). A publicly availableWWW server (http://shgc-www.stanford.edu) permitted chromosomallocalization of markers. The PCR amplification with the same set ofprimers was set up in a 96-well microtiter plate and used in aRoboCycler Gradient 96 thermal cycler (Stratagene). The PCR productswere separated by electrophoresis on a 2% agarose gel.

The PAR4 gene was mapped to chromosomal location 19p12. This locationwas different from that of the PAR1 and PAR2 genes, which are locatedwithin approximately 100 kb of each other at chromosome 5q13. Thelocation of the two latter genes suggested that they arose from a geneduplication event (M. Kahn et al., Mol. Med. 2:349–57, 1996). Atpresent, the localization of PAR3 is unknown. Additional members of thePAR family probably exist that have evolved through a combination ofretroposition and gene duplication (W. C. Probst et al., DNA Cell Biol.11:1–20, 1997).

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An isolated polynucleotide molecule that encodes a polypeptidecomprising amino acid residues 48 to 78 of SEQ ID NO:2.
 2. The isolatedpolynucleotide molecule of claim 1, wherein the polynucleotide moleculecomprises nucleotides 317 to 409 of SEQ ID NO:1.
 3. A compositioncomprising the isolated polynucleotide molecule of claim
 1. 4. Theisolated polynucleotide molecule of claim 1, wherein the polynucleotidemolecule is a DNA molecule.
 5. A vector, comprising the isolatedpolynucleotide molecule of claim
 1. 6. The isolated polynucleotidemolecule of claim 1, wherein the polynucleotide molecule encodes apolypeptide comprising amino acid residues 18 to 78 of SEQ ID NO:2. 7.The isolated polynucleotide molecule of claim 6, wherein thepolynucleotide molecule comprises nucleotides 227 to 409 of SEQ ID NO:1.8. The isolated polynucleotide molecule of claim 1, wherein thepolynucleotide molecule encodes a polypeptide comprising amino acidresidues 18 to 385 of SE ID NO:2.
 9. The isolated polynucleotidemolecule of claim 8, wherein the polynucleotide molecule comprisesnucleotides 227 to 1330 of SEQ ID NO:1.
 10. The isolated polynucleotidemolecule of claim 1, wherein the polynucleotide molecule encodes apolypeptide comprising amino acid residues 1 to 385 of SEQ ID NO:2. 11.The isolated polynucleotide molecule of claim 10, wherein thepolynucleotide molecule comprises nucleotides 176 to 1330 of SEQ IDNO:1.
 12. An isolated polynucleotide molecule that encodes the aminoacid sequence of SEQ ID NO:7.
 13. An isolated polynucleotide moleculecomprising a nucleotide sequence that is complementary to the nucleotidesequence of SEQ ID NO:1.
 14. An expression vector, comprising apolynucleotide molecule that encodes a polypeptide comprising amino acidresidues 18 to 78 of SEQ ID NO:2, a transcription promoter, and atranscription terminator, wherein the promoter is operably linked withthe polynucleotide molecule, and wherein the polynucleotide molecule isoperably linked with the transcription terminator.
 15. The expressionvector of claim 14, wherein the polynucleotide molecule encodes apolypeptide comprising amino acid residues 18 to 385 of SEQ ID NO:2. 16.A host cell comprising the expression vector of claim 14, wherein thehost cell is selected from the group consisting of bacterium, yeastcell, fungal cell, insect cell, mammalian cell, and plant cell.
 17. Thehost cell of claim 16, wherein the host cell is a bacterial host cell,which is either an E. coli cell or a Bacillus cell.
 18. The host cell ofclaim 16, wherein the host cell is a fungal cell, which is either aSaccharomyces cell or a Pichia cell.
 19. The host cell of claim 16,wherein the host cell is a mammalian cell.
 20. The host cell of claim19, wherein the mammalian cell is a human cell.
 21. A method of usingthe expression vector of claim 14 to produce a protease activatedreceptor-4 polypeptide, the method comprising the act of culturing hostcells that comprise the expression vector and that produce a proteaseactivated receptor-4 polypeptide.
 22. The method of claim 21, furthercomprising the act of isolating the protease activated receptor-4 fromthe cultured host cells.
 23. A virus, comprising the expression vectorof claim 14.